1. Field of the Invention
The present invention relates to a microelectrode, and a microelectrode array of which temperature can be controlled in a solution, and in particular, to the microelectrode and the microelectrode array which have excellent thermal isolation between a microheater and a substrate, which has a small power consumption for heating, which has high heating and cooling speed and which can be manufactured by semiconductor manufacturing process.
2. Disclosure Statement
Presently, researches have been vigorously performed about the microelectrode of which the potential of the electrode can be adjusted in a state where the microelectrode is dipped in a solution. Such a microelectrode can not only be used as a measuring electrode of an electrochemical sensor, but also be used in site-selectively obtaining a micropattern or in controlling an interaction or transport of a biomolecule by controlling the potential of the microelectrode.
Now, review will be done on concrete examples in which the microelectrode is applied to the electrochemical measurement, micropatterning and control of the biomolecule.
As interest in personal health and environment has been increased, development of small sensors which can obtain various precise information in short period of time is required. In particular, development of sensors using the microelectrode and electrochemical measurement method is required to miniaturize these sensors. As electrochemical sensors using the microelectrode, there are electrochemical biosensor using an enzyme and a potentiometric sensor which measures pH or concentration of ions from a potential difference. Recently, electrochemical DNA sensors and electrochemical immunosensors using the microelectrode are manufactured, too.
The electrode array in which there are many number of microelectrodes in a substrate are applied to an electrochemical DNA chip, an electrochemical protein chip and an electronic tongue, etc. in which multiple processes can be performed parallel.
In addition, the microelectrode composed of interdigitated array (IDA) structure can be used in measuring a change of electrode surface from impedance or in improving sensitivity by amplifying the electrode reaction. In case of bonding or integrating the microelectrode with fluid control devices, it can be possible to construct a lab-on-a-chip in which separation, reaction and detection can be performed in a single chip.
Generally, micropattern can be manufactured by the semiconductor manufacturing process such as deposition, photolithography and etching, or by electrodeposition. In case of using the electrodeposition process, not only metals such as copper but also conducting polymer and metal oxide having electric conductivity can be site-selectively deposited. If electrodeposition is performed on the microelectrode, the micropattern of the metal, polymer and metal oxide can be obtained with ease.
Recently, many attempts have been trying to site-selectively immobilize the biomolecule on the electrode by voltage control (Cosnier, Serge, “Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review” Biosensors & Bioelectronics 14: pp. 443-456 (1999)). A method of site-selectively electropolymerizing biomolecule-attached monomer on the microelectrdode, or site-selectively forming the biomolecule-entrapped polymer on the microelectrode in a solution containing monomer and biomolecule has been developed. Also developed is a method by Nanogen Company in which DNA immobilization is controlled by electrode potential (refer to U.S. Pat. No. 5,605,662). Since DNA has negative(−) charge, it moves to the electrode if positive voltage is applied to the electrode. At this time, the moved DNA is immobilized on the electrode surface. Eventually, DNA is site-selectively immobilized on the electrode by potential control.
In addition, a method of site-selectively immobilizing the biomolecule by electrochemically changing the pH around the electrode has been developed. Combimetrix Company suggested a method of site-selectively synthesizing an oligonucleotide to the microelectrode by using such concept. (U.S. Pat. No. 6,093,302).
Since the biomolecule such as DNA or protein has multiple electric charges, the transport or interaction of DNA and protein can be controlled by potential control. Nanogen Company has been accomplished that positive potential is applied to the microelectrode immobilized with single-strand DNA so that the single-strand DNA which is complementary to the DNA is hybridized in short time(U.S. Pat. No. 5,849,486). DNA in which bases of two single-strand DNA are not completely complement is dehybridized by applying the negative potential to the microelectrode (U.S. Pat. No. 6,017,696).
As described above, electrochemical measurement of biomolecule, micropatterning of biomolecule and control of biomolecule can be done by potential control of the microelectrode. However, to manufacture an electrode having better performance, the potential control of the electrode as well as temperature control is necessary. A research on the electrode of which temperature control is possible is mainly performed by Gründler Group (Gründler, Peter, et al., “The technology of hot-wire electrochemistry”, Electroanalysis 11: pp. 223-228(1999)). It is expected that the micropatterning of biomolecule and the control of biomolecule in addition to the electrochemical measurement of biomolecule will be possible by adjusting the temperature of electrode in the same way as in the case of the potential control.
In terms of electrochemistry, when temperature of the electrode is varied, the electrochemical kinetics, mass transport and electrochemical thermodynamics are influenced. The electrochemical kinetics increases exponentially with temperature, and the transport of material around electrode occurs vigorously due to heat convection. Therefore, the current increases according to the increase in temperature, which can be used in raising the sensitivity of an electrochemical sensor. If the temperature changes, redox potential associated with electrochemical thermodynamics changes too, and the temperature changes of the electrode surface can be indirectly measured from the changes of the redox potential.
The reaction and interaction control of biomolecule is possible to some degree by adjusting the temperature because biomolecule is very sensitive to temperature. The activity of biomolecule such as enzyme increases with temperature. Since the activity of biomolecule can be easily lost if the high temperature is maintained for long period, therefore, appropriate temperature control is necessary according to each biomolecule. An example of the control of biomolecule using temperature control is adjustment of DNA hybridization/dehybridization. When the temperature rises, the dehybridization of DNA occurs, and when the temperature falls, the hybridization of DNA occurs again. If dehybridization/hybridization is repeated while cloning the DNA of desired part at the time of dehybridization, it is possible to amplify DNA in great amount in short period. At this time, precise temperature adjustment and rapid heating and cooling are indispensable for rapid and precise amplification.
If the temperature control of the electrode is possible when the biomolecule is immobilized on the electrode surface, the biomolecule can be effectively controlled. The redox reaction and the micropatterning are highly influenced by temperature of the electrode surface. Therefore, it is not necessary to heat up the solution entirely but only to heat up the electrode surface for such control. It is effective only to heat the electrode surface or the surroundings of the electrode for rapid heating and cooling. Even if the electrode is only heated, if the volume of the solution is extremely small, the temperature control of the solution is possible by heating the microelectrode alone.
If a large amount of electric power is consumed in adjusting the temperature, a large battery or power source is required even if the volume of the electrode and measurement circuit is small. Eventually, entire size of the small measurement system depends on the size of battery or power source. Therefore, to manufacture the small sensor, the temperature-controllable electrode with small power consumption must be used.
Since the size of the microelectrode can be decreased to a very small size if the semiconductor manufacturing process is used, the electric power required to increase the temperature of the electrode surface is not large. Even in a solution of large heat capacity, if the size of the electrode is small, the electrode surface can be rapidly heated. Also, since the convection layer of the solution caused by the heat is not large, when the heating is interrupted, the temperature of the electrode falls down to the temperature of surroundings in short period.
As described above, the temperature control of the electrode can be applied to electrochemical measurement and control of biomolecule, and for this, it is required above all to use the microelectrode of precise temperature control and high speed of heating and cooling. Now, conventional technologies for adjusting the temperature of the electrode will be reviewed below.
The mostly frequently used method in adjusting the temperature of the electrode is a method in which the solution is entirely heated or cooled thereby raising or lowering the temperature of the electrode. However, this method has many disadvantages that since in that method the solution is entirely heated, it takes long time to raise or lower temperature, and it is difficult to maintain entirely the temperature of the solution constant.
There is also a method in which the electrode is heated by radiation. One example is to instantaneously heat the electrode by shooting the laser to the front surface or back surface of the electrode. This method is frequently used in checking the change with time after instantly heating the electrode. However, this method has the problems that it is difficult to maintain the temperature of the electrode and the device is expensive and the entire volume is large since laser is used. There is also a method of heating the electrode with the light produced from the tungsten lamp. However, this method has difficulty in efficiently adjusting the temperature, too.
Recently, a method of directly heating the electrode by Joule heating is also used (Gründler, Peter, et al., “The technology of hot-wire electrochemistry”, Electoanalysis 11: pp. 223-228 (1999)). This is a method of heating the electrode by applying high frequency alternating current of about 100 kHz to the electrode. However, this method has problem that electric power consumption is large, and the redox current can be influenced by the alternating current. Furthermore, there are problems that since the alternating current of high frequency is used, an expensive alternating current generator is necessary, and large power is consumed. In addition, since the resistance of the wiring which connects the electrode to the external circuit is larger than that of the electrode in case of manufacturing the microelectrode in silicon substrate, there is a great possibility that most of electric power is consumed in the wiring than in the electrode. Therefore, this method can not be used in manufacture of the microelectrode.
Heating by RF(radio frequency) radiation is reported as a method of heating by inducing eddy current to the metal electrode, however, this method has problem that electric power consumption is serious and miniaturization is difficult, too (Qiu, Fulian, et al. “Thermal activation of electrochemical processes in a RF-heated channel flow cell: experiment and finite element simulation”, Journal of Electroanalytical Chemistry 491: pp. 150-155 (2000)).
A method of indirectly heating the electrode by a microheater is used too. In this method, heat generated from Joule heating in the microheater is transferred to the electrode through a medium to heat the electrode. Since voltage or current must be applied to the microheater for Joule heating, the microheater must be electrically insulated from the electrode. To make the heat generated from Joule heating to be effectively transferred to the electrode, there must be no material of high heat conductivity around the microheater. A silicon substrate of very high heat conductivity is mainly used for semiconductor device manufacturing. In case where the microheater exists on the silicon substrate, substantial amount of heat generated at the microheater is not transferred to the electrode but leaks out to the silicon substrate. To reduce such heat loss, a method of forming an etched pit on the silicon substrate around the microheater or forming a cavity between silicon substrate and the heater is used.
Indirect heating of the microelectrode with the microheater is substantially effective in view of power consumption or applicability to a sensor among the methods of adjusting the temperature of the electrode as described above. Formation of the etched pit or the cavity for reducing heat loss of the microheater can be implemented by micromachining technology. The micromachining technology can be classified into bulk micromachining for machining upper and lower surfaces of the silicon substrate and surface machining for stacking a thin film on top of the substrate, etching and machining the thin film.
The bulk micromachining is a method of removing silicon from the surroundings of the microheater by etching from a defined area of upper or lower surface. Electric heat loss can be substantially decreased by making a structure of bridge, cantilever and membrane separated from the substrate by forming the etched pit or cavity on the substrate, by using such a method and thereafter forming the microheater. In this case, the manufacturing is easy, however, there is a limit in decreasing the heat loss since it can not essentially remove the air existing in the etched pit or cavity. In addition, the method has a disadvantage that the structure cannot be manufactured with the standard CMOS process.
The surface micromachining is a method of forming a microcavity by etching a sacrificial layer formed on a top surface, and forming the microheater on the cavity. This method has an advantage of manufacturing with the standard CMOS process, and making a microcavity array with ease. However, there is no report so far of the microelectrode in which the temperature can be adjusted in solvent by using the surface micromachining.
When using the microelectrode having a cavity in solution, there is a problem that the solution enters into the cavity so that the electric heat loss of the microheater becomes large. Therefore, the cavity of the microelectrode to be used in the solution must be sealed. In case this cavity is sealed under vacuum, the heat loss of the microheater becomes very small.
In a method of manufacturing a structure having a cavity on a silicon substrate by using the surface micromachining, conventional technology status disclosed as references or patents will be reviewed below.
First, U.S. Pat. No. 6,023,091 discloses a structure in which a cavity and microheater are formed on a silicon substrate, where the cavity is formed by depositing and etching a sacrificial layer without etching the silicon substrate. The manufacturing process is simple, however, there is a disadvantage that effective thermal isolation cannot be obtained since the depth of cavity cannot be made large.
In U.S. Pat. No. 5,948,361, a substrate A formed with microheater is junctioned to a substrate B formed with cavity, and then the substrate A is removed leaving the microheater. This method has a problem that it is difficult to align and junction the two substrates.
U.S. Pat. No. 5,907,765 discloses a method comprising the steps of: forming an insulation film on a silicon substrate; etching the silicon substrate in a portion to be used as a cavity; filling a sacrificial layer; forming a heater film; etching the sacrificial layer through an etching channel; and sealing the etching channel. There is a problem that in the step of filling the sacrificial layer after etching the silicon substrate, it is difficult to form the sacrificial layer thick and to a desired shape. That is, a sealed cavity with excellent thermal isolation performance cannot be obtained.
U.S. Pat. No. 5,296,408 discloses a method comprising the steps of: etching a silicon substrate; filling aluminum, depositing a silicon oxide film SiO2; and producing a sealed cavity in the place where aluminum was by diffusing the aluminum with heat.
Reference (Liu, Chang et al., “Sealing of micromachined cavities using chemical vapor deposition methods: characterization and optimization” 8: pp. 135-145 (1999)) discloses a method comprising the steps of: defining a silicon nitride film Si3N4 on the silicon surface; forming a thermal oxide sacrificial layer by thermal oxidizing the surface of a portion where silicon is exposed; forming a sacrificial layer to be used as etching path; forming a supporting film from the silicon nitride film, forming a cavity by forming an etching hole and thereafter etching the sacrificial layer; and sealing the etching hole under vacuum. Since the cavity is vacuum-sealed cavity, in case where the microheater exists on the sealed cavity, it can obtain good thermal isolation. However, since the depth of sealed cavity has to be made within 1 to 2 μm, there is a limit in making the thermal isolation performance excellent.
A good deal of researches on formation of an efficient sealed cavity has been performed as seen in the patent and references stated above, however, a good deal of rooms are left to improve the thermal isolation. In particular, a development of sealed cavity is necessary which can have excellent thermal isolation performance by making the depth of sealed cavity large.
On the other hand, in case of microelectrode which is used in solution and heated indirectly with microheater, following problems exist in insulation between a microheater and an electrode, corrosion of wirings, and resistance of wirings as well as a sealed cavity with good thermal isolation.
In case of heating the electrode with heat produced by applying voltage or current to microheater, and measuring the redox current flowing to the electrode, the voltage or current of the microheater can influence current of the electrode. For example, if the difference of voltages each applied to the microheater and the electrode is 1 V, and resistance of an insulation film between the microheater and the electrode is 1 GΩ, then, current of 1 nA can flow between the microheater and the electrode. Since this amount of current can influence the redox current, the insulation performance of the insulation film must be very good. In particular, the insulation performance of the insulation film is far more important when applying high voltage to the microheater to increase the temperature of the electrode.
Corrosion of wirings can be another problem in the microelectrode which can adjust the temperature in solution. There are many cases where the solution in which the microelectrode is to be dipped has high concentration ion (especially, chlorine ion), in general, aluminum used in wirings in semiconductor manufacturing process is corroded with ease in the solution. The corrosion can be reduced substantially if a protection film is formed on metal wirings, however, in case there are many pin holes in the protection film, the corrosion of metal wirings can easily arise. Since the protection film consisting of a silicon oxide film or a silicon nitride film is formed with PECVD (low pressure chemical vapor deposition) in the low temperature under the condition that the metal wirings are formed, there are many pin holes in the film.
Voltage or current is applied to heat the microheater, where if the voltage or current applied to resistor changes, temperature of the electrode changes. The resistance of microheater must be uniform to have same temperature performance regardless of the electrode when same voltage is applied, and resistance of wirings connecting the microheater must be very small in comparison to that of the microheater. Since the number of necessary wirings and the area of wirings are increased in case of making a microelectrode array, the line width of wirings must be decreased. Since the resistance of wirings increases if the line width is decreased, metal of low specific resistivity must be used to lower the resistance of wirings. Generally, aluminum widely used as wirings has large specific resistivity and a thick film can be deposited with aluminum with inexpensive cost, and a micropattern can be obtained with aluminum, however, there is a disadvantage that aluminum is corroded easily in solution. Another metal that can be used as wirings in a CMOS process is platinum, however, since platinum has high specific resistivity, a platinum film must be formed thick to reduce the resistance. In this case, it costs high expense and it is difficult to form micropattern with dry or wet etching. Therefore, there is a limit in using the platinum as wirings of a microheater. In case of gold, although it has low specific resistivity, since the standard CMOS process cannot be performed after deposition, it cannot be used as wirings. Metals such as silver, copper, etc. having small specific resistivity are not easily used in general semiconductor manufacturing process.
It is required to manufacture a microelectrode having adjustable temperature in the solution to apply the microelectrode to electrochemical measurement of biomolecule, micropatterning of biomolecule and control of biomolecule as described above.